SK280619B6 - Preparation eliciting a protective immune response to helicobacter infection, a vaccine and method of evaluating in a mammalian host endogenous immune response - Google Patents

Abstract

Preparation for eliciting in a mammalian host a protective immune response to Helicobacter infection by administering to the host an immunogenically effective amount of a Helicobacter urease or urease subunits as antigen.

Description

The composition for inducing a protective or therapeutic immune response to an infection caused by Helicobacter in a mammalian host comprises an immunologically effective amount of urease originating from Helicobacter or its subunits as an antigen.

Technical field

The invention relates to a suitable vaccine which is useful for the prevention and treatment of infection caused by Helicobacter bacteria in mammals, including humans.

BACKGROUND OF THE INVENTION

Helicobater bacteria that cause gastritis in human gastric epithelial infection are a major factor in the development of gastrointestinal ulcers and gastric lymphomas, and may also be a risk factor in the development of gastric cancer [Blaser, Gastroenterology 1987, 93, 371-383; Graham, Gastroenterology 1989, 196,615-625; Parsonnet, J. Natl. Cancer Inst. 1991, 93, 640-643]. The most common infectious agent is Helicobacter pylori, followed by the much less prevalent Helicobacter heilmanii. Helicobacter pylori is a narrow, gram-negative, S-shaped microorganism that is routinely obtained from biopsies of stomachs of adults and children with histological signs of gastritis or ulcers of the digestive tract. Evidence of a causal relationship between the occurrence of Helicobacter pylori and gastroduodenal diseases comes from studies in volunteers, patients with ulcers and stomach cancer, gnotobiotic pigs and rodent-free germs. With regard to etiology, Koch's postulates have been met by the development of histologically confirmed gastritis in previously uninfected individuals as a result of the consumption of living microorganisms [Marshall, Med. J. Aust. 1985, 142, 436-439; Morris, Am. J. Gastroenterology, 1987, 82,192-199; Engstrand, Infect. Immun. 1990, 53, 1763-1768; Fox, Infect. Immun. 1988, 56, 2994-2996; Fox, Gastroenterology 1990; 99: 352-361; Lee, Gastroenterology 1990, 99, 1315-1323; Fox, Infect. Immun. 1991, 59, 785-791; Eaton, Infect. Immun. 1989, 57, 1119-1125], Gastritis disappeared after treatment that eliminated Helicobacter pylori and relapse rates in patients with gastric ulcers [Peterson, N. Engl. J. Med. 1991, 324, 1043-1048],

It is often very difficult to eradicate a developed infection of Helicobacter pylori with antimicrobial agents in vivo, despite their susceptibility to many antimicrobial agents in vitro [Czinn, Infect. Immun. 1991, 2359-2363], The microorganism is found in the mucosal layer covering the gastric epithelium and in the gastric lobes. Adequate antimicrobial concentrations of the drug cannot be achieved at these sites, even when antibiotics are administered orally and at high doses. Currently, most authorities recommend 'triple therapy', especially bismuth salt in combination with drugs such as tetracycline and metronidazole for two to four weeks. However, the efficacy of this or other chemotherapeutic route of administration remains less than optimal. In addition, this treatment may cause serious adverse reactions to the administered drugs.

At present, little is known about the role of the mucosal immune system in the stomach. The distribution of immunoglobulin (Ig) producing cells in the normal gastric cavity shows that IgA-producing plasma cells account for more than 80% of the total plasma cell population. In addition, the amount of plasma IgA cells present in the gastric cavity are comparable to other mucous membranes [Brandtzaeg, Scand. J. Immunol. 1985, 22, 111-146; Brandtzaeg, Ann. Allergy 1987, 59, 21-39]. Numerous human studies [Wyatt, J. Clin. Path. 1986, 39, 863-870] and animal models [Fox, Gastroenterology 1990, 99, 353-361; Fox, Infect. Immun. 1991, 59, 785-791] shows a specific IgG and IgA response in serum and gastric secretions in response to Helicobacter infection. Observations suggest that Helicobacter pylori infection persists as a chronic infection for several years and, despite induction of a local and systemic immune response, does not support the development of immunization strategies.

Lee et al. have described the ability of Helicohacter felis (closely related to Helicobacter pylori) to infect rodents that have not previously been infected, and reproducibly documented histological gastritis [Lee, Gastroenterology 1990, 99, 1315-1323; Fox, Infect. Immun. 1991, 59, 785-791]. Hence, this bacterial-host pair is accepted as a good model for the study of gastritis caused by Helicobacter bacteria and their initiation factors [Lee, Infect. Immun. 1993, 61, 1601-1610]. Czinn et al. have shown that repeated oral immunization with crude juice from lysed Helicobacter pylori bacteria in which choleratoxin was used as an adjuvant indicates a strong gastrointestinal IgA response with anli-Hehcobacter pylori specificity in mice and lacs [Czinn, Infect. Immun. 1991, 2359-2363], Further, Chen et al. and Czinn et al. recently reported that oral immunization with crude juice from lysed Helicobacter felis bacteria provides protection against Helicobacter felis infection in mice [Chcn, Lancct, 1992, 339, 1120-1121; Czinn, American Gastroenterological Association. May 10-13, 1992, 1321, A-331]. The exact nature of the antigen (s) responsible for inducing this protection has not yet been established, and no information suggests that the protective antigen (s) from Helicobacter felis that caused protection against this pathogen could induce cross-protection for other species of bacteria Helicobacter.

According to the invention, it has been found that Helicobacter pylori and Helicobacter felis sonicates are capable of inducing antibody formation, showing that some of these antibodies directed against Helicobacter pylori are capable of cross-reacting with Helicobacter felis and vice versa [Michetti, American Gastroenterology Association. May 10-13, 1992, 1001, A-251; Davin, American Gastroenterological Association, May 16-19, 1993, 1213, A-304]. The nature of these cross-reactivities was unknown.

Based on the existing homology between the various known urease amino acid sequences, it has been suggested that urease could be used as a vaccine against Helicobacter pylori [Pallen, Lancet 1990, 336, 186-7]. Nevertheless, cross-reactivity is not the rule. Guo and Liu years ago have shown that urease from Proteus mirabilis, Proteus vulgaris and Providencia rettgeri have cross-reactivity to each other, while ureas from Canavalia (jack bean) and Morganella morganii are immunologically distant from the three ureas mentioned above [Guo, J. Gen. Microb. 1965,136 1995-2000]. Although the possibility of antigenic cross-reactivity of Helicobacter pylori urease with ureases of other helicobacteria was a reasonable presumption, there was no evidence to show that this is the true cause of cross-reactivities unless the invention has shown that some monoclonal antibodies against Helicobacter felis have reacted with Helicobacter urease Davin, American Gastroenterology Association, May 16-19, 1933, 1213, A-304], J. Pappo further demonstrated that mice that were infected with Helicobacter felis bacteria produced antibodies capable of cross-reacting with Helicobacter pylori urease, but did not react with urease from Canavalia (J. Pappo, unpublished data, 1993).

The use of Helicobacter pylori urease or related urease as a vaccine against Helicobacter pylori infection has been suggested earlier by A. Labign [patent application

SK 280619 Β6

EPO EP 0 367 644 A1, 1989, Int. Publication No. WO90 / 04030, 1990], but the present application contains no evidence of vaccination of any mammal against any helicobacterial infection by urease.

In addition, while sequence homology with other bacterial ureases may support the use of urease as a suitable vaccine candidate for Helicobacter pylori infection, current knowledge of the course of human Helicobacter pylori infection will certainly not support it. Although infected individuals often produce a strong antibody response against urease, this anti-urease immune response does not lead to destruction or control of the infection. In addition, Helicobacter pylori is able to transport urease out of the cell and release it from its surface [Evans, Infect. 1992, 60, 2125-2127; Ferrero, Microb. Ecol. Health Dis. 1991, 4, 1120-1121]. Therefore, urease cannot represent a suitable target for the development of a protective mucosal immune response. Indeed, it is generally believed that mucosal immune protection is largely mediated by secreted IgA whose agglutination activity deteriorates when the recognized antigen can be released from the target pathogen and thus serves as a delusive target for the protective antibody. Furthermore, urease appears to be toxic to epithelial cells in culture, and is suspected to play a role in mucosal degradation and in the development of gastrointestinal ulcers in vivo. Therefore, its use as an antigen may be toxic.

However, it can be concluded that this antigen could be a potentially effective vaccine if:

- first, the antigen could be administered orally so that the dose is high enough to induce a stronger immune response than is naturally occurring

- secondly, the amount of antibodies produced must be sufficiently high to bind all urease, both released and not released from the cell surface

thirdly, if we could use urease subunits or non-toxic molecular variants.

Finally, there remains a need for effective treatment and prevention of gastric human infections caused by Helicobacter pylori. Recent data suggested the possibility to create a vaccine against this infection, but did not clearly identify the defined antigen (s) common to all Helicobacter pylori strains that could be incorporated into a safe and effective vaccine.

According to the invention, a Helicobacter pylori urease antigen is identified as a possible vaccine and its efficacy is demonstrated in an animal model. These results are unexpected in the context of the history of Helicobacter infections.

SUMMARY OF THE INVENTION

According to the present invention, it has been found that immunity can be induced in mammals susceptible to gastrointestinal Helicobacter infection when using urease epitopes occurring on or near the surface of Helicobacter as vaccine target sites. Either natural urease or recombinant urease subunit, which is produced in enzymatically inactive and therefore non-toxic form, can be used to induce immunity. The invention discloses a method of inducing immunity against Helicobacter infection by administering a polyamino acid preparation to a mammalian mucosal surface, i. j. a mixture of peptides and / or proteins, together with a suitable adjuvant. A plurality of epitopes characteristic and presented by the urease endogenous to a bacterium of the genus Helicobacter to be infected are present in this polyamino acid preparation. The polyamino acid preparation can be administered orally.

The active ingredient of the formulation may contain natural or biosynthetic epitopes and may take various forms. Countless possible preparations include purified, naturally occurring or recombinantly produced urease preparations of bacterial or other origin, proteolytic urease fragments, fusion proteins containing urease epitopes, altered urease forms, or peptides homologous to the urease amino acid sequence. Because the induction of immunity depends on the induction of a humoral and / or cellular immune response that is directed against the infecting Helicobacter, those compositions that best duplicate epitopes of endogenous urease of the infecting organism are preferred. For example, preparations containing Helicobacter pylori urease epitopes are preferably administered to people susceptible to Helicobacter pylori. In addition, with respect to an important aspect of the invention, it has been found that urease from other species may be used. For example, it has been found that the prevention of Helicobacter felis infection in mice may occur after administration of Helicobacter pylori urease.

It is an object of the invention to provide a protective immune response in a mammalian host against Helicobacter infection, characterized in that an immunologically effective amount of urease capable of eliciting such a protective immune response is administered to the mucosal host surface.

The invention further provides a vaccine composition suitable for preventing Helicobacter infection and comprising an effective amount of urease as an antigen, preferably a Helicobacter pylori urease or a B subunit of Helicobacter pylori urease, capable of eliciting a protective immune response in a host against Helicobacter infection with a Helicobacter or a solvent.

The invention further provides a method of imparting passive protection to a mammalian host against Helicobacter infection, comprising administering an immunologically effective amount of a specific urease antibody to the mucosal surface of the host. This antibody is produced by a host immunized with urease, preferably Helicobacter pylori urease or the B subunit of Helicobacter pylori urease, and is capable of eliciting a protective immune response against Helicobacter infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described with reference to the figures, wherein Figures 1 to 6 are a graphical representation of the results arranged in Tables 1 to 6.

Figure 1 (Group A): Mice not protected after urease immunization.

X axis: "o" denotes IgG, "o" denotes IgA.

The Y-axis shows the immunoglobulin concentration measured spectrophotometrically. The figure corresponds to the absorbance at 495 nm multiplied by 1000.

Figure 2 (Group B): Mice protected after urease immunization.

X axis: "o" denotes IgG, "o" denotes IgA.

The Y-axis shows the immunoglobulin concentration measured spectrophotometrically. The figure corresponds to the absorbance at 495 nm multiplied by 1000.

Figure 3 (Group C): Mice not protected after immunization with Helicobacter pylori sonicate.

X axis: "o" denotes IgG, "o" denotes IgA.

The Y-axis shows the immunoglobulin concentration measured spectrophotometrically. The figure corresponds to the absorbance at 495 nm multiplied by 1000.

Figure 4 (Group D): Mice protected after immunization with Helicobacter pylori sonicate.

The X-axis shows: "o" denotes IgG, "o" denotes IgA.

The Y-axis shows the immunoglobulin concentration measured spectrophotometrically. The figure corresponds to the absorbance at 495 nm multiplied by 1000.

Figure 5: Shows the percentage of mice that developed infection (Y axis).

X axis A: A group of mice immunized with Helicobacter pylori sonicate. Infection did not develop in three of the nine experimental animals.

B: Group of mice immunized with urease Helicobacter pylori. Infection did not develop in seven out of ten immunized animals.

C: Control group of mice. Infection did not develop in one of ten experimental animals (p = 0.019 in Fisher's precision test).

Figure 6: summarizes the results obtained after oral immunization with recombinant A and B urease subunits. On the X-axis, the number indicates the number of test animals. In the rectangle indicated by the symbol I, control mice were not protected by immunization. In the rectangle marked with the symbol II, animals are protected by immunization with recombinant A (upper half of the figure) and B (lower half of the figure) urease which were sacrificed 12 days after exposure to the pathogen, while animals in the rectangle marked III were sacrificed after 10 weeks but otherwise treated the same way. Fisher's precision values: p = 0.003 (mice immunized with urease B subunit after 12 days) and p = 0.01 (immunization with B urease B subunit after 10 weeks).

The values on the Y-axis indicate the amount of absorbance at 550 nm.

According to the invention, it has been found that oral administration of polyamino acid preparations presenting Helicobacter pylori urease epitopes causes an increase in the protective immune response against Helicobacter felis in mice. The mouse is used as an animal model of generally accepted value to study the immune response to Helicobacter infection [Lee, Gastroenterology 1990, 99, 1315-1323]. The effect of such a protective immune response is that immunized animals, when exposed to a pathogen, have very reduced incidence of infection compared to non-immunized animals. Oral immunization of mice with the Helicobacter pylori urease B subunit, which is produced as an enzymatic inactive recombinant protein, results in an increase in the protective immune response against Helicobacter felis in mice. The effect of this protective immune response is that immunized animals, when exposed to the pathogen, have a very reduced incidence of infection, as compared to non-immunized animals.

The invention provides a method of inducing a protective immune response to Helicobacter infection in a mammalian host. The method comprises the step of administering an immunologically effective amount of urease as an antigen, preferably a Helicobacter pylori urease, capable of eliciting such a protective immune response to a mucosal surface of a mammal, including a human.

The invention further provides a method of inducing a protective immune response to Helicobacter infection in a mammalian host. The method comprises the step of administering an immunologically effective amount of a recombinant, enzymatically inactive B subunit urease as an antigen, preferably a recombinant B subunit urease from Helicobacter pylo ri, capable of eliciting such a protective immune response to a mucosal surface of a mammal, including a human.

The invention also relates to the treatment or preventive protection of a mammal, including a human, from Helicobacter infection, wherein an immunologically effective amount of urease or their subunits capable of eliciting a protective immune response to Helicobacter infection is administered to the mucosal surface of the patient. Preferably, a uricase from Helicobacter pylori or the B subunit of a uricase of Helicobacter pylori is used, and the urease may be administered either alone or coupled to a hydroxylated calcium phosphate, for example hydroxyapatite, which serves as a carrier particle. It is further preferred to administer the urease of Helicobacter pylori together with a mucosal adjuvant, for example with the B subunit of choleratoxin, muramyl dipeptide and / or other similar adjuvants.

It is believed that administration of urease or its B subunit as an antigen to the mucosal surface stimulates the overall mucosal immune system and perhaps local sites in the gastric mucus, including the immune response, including the appearance of specific Helicobacter pylori IgA antibodies in gastric secretions Helicobacter infection. Since it is a routine matter to conduct preclinical tests of future vaccines for human use in animal models, it can be concluded that the methodology employed in this invention will be effective in humans, particularly in the prevention and treatment of gastrointestinal ulcers, gastritis, gastric malignancies and other diseases, resulting from the presence of Helicobacter pylori and / or Helicobacter heilmanii.

A-Bacterial cultures and urease purification

The Helicobacter pylori strain used for the study came from a patient suffering from duodenal ulcer and was pre-grown on BHI agarose plates so that the resulting culture was homogeneous. Helicobacter pylori is grown on a suitable medium, typically BHI medium (Brain-Heart Infusion; medium containing heart and brain extract) containing 0.25% yeast extract and 10% fetal calf serum and supplemented with 0.4% supplement for selective growth of Campylobacteria. (supplemented by Skirrow; Oxoid 69). The bacteria are incubated overnight under microaerophilic conditions at 37 ° C in bottles, which are then sealed and shaken at 37 ° C for two to three days to form a liquid culture. The culture can also be prepared on agarose plates containing BHI medium with 0.25% yeast extract and 5% sheep blood. Cultivation takes place under microaerophilic conditions at 37 ° C for three days. The amount of bacteria is determined by measuring the absorbance of the BHI solution, at a wavelength of 660 nm, one absorbance unit corresponding to 10 ⁸ bacteria. Cultures on agarose plates are first resuspended in 154 mM NaCl.

A common and preferred source of polyamino acid having urease epitopes is purified urease, for example, Helicobacter pylori urease, obtained by the general method of Dunn et al., J. Biol. Chem. 265, 9464-9465. This method is modified as described below. After cultivation, the cells are lysed in water, centrifuged, then the sediment is vortexed and centrifuged again to form a supernatant. The solution having Helicobacter pylori urease activity (estimated by a rapid urease test, see below) is then subjected to chromatography on a CL-6B column. Fractions having strong urease activity were collected and dialyzed overnight, then re-subjected to anion exchange chromatography. Fractions were eluted with increasing NaCl buffer and the collected fractions with strong urease activity were subjected to SDS gel electrophoresis followed by Coomassie blue staining. Two separate bands corresponding to a molecular weight of about 63 kDa and about 29 kDa have been identified as urease. Fractions containing urease are pooled to give purified Helicobacter pylori urease with a purity of 95% to 99%.

B-Oral immunization with urease from Helicobacter pylori

While it is preferred to use a urease purified from Helicobacter pylori obtained as described above, it is understood that any urease or urease subunit, either naturally occurring or obtained by recombinant DNA techniques, as well as a proteolytic fragment, fusion may be used as the antigenic material. proteins containing fragments or whole urease, altered urease constructs, or other peptides or protein preparations containing urease epitopes capable of eliciting a protective immune response against Helicobacter infection (see below). Thus, a urease having partial homology with respect to Helicobacter pylori urease and which is effective in eliciting a cross-protective immune response against Helicobacter can be used. An example of such a urease is Canavalia urease which has about 70% homology to Helicobacter pylori urease. Therefore, the invention is not limited to the use of intact urease, but encompasses the use of any polyamino acid preparation that contains urease epitopes and is capable of eliciting a protective immune response in the host against Helicobacter infection. Typically, urease having a homology in the range of 70 to 95%, for example 80% to 90% homology, to urease from Helicobacter pylori, may be used as the antigen urease of the invention.

Countless sources of potentially useful urease preparations include endogenous urease enzymes of different Helicobacter species, urease from other bacteria such as Klebsiella pneumoniae or Proteus mirabilis and, analogously, any other urease, provided that the urease has a uricase reaction with uricobacterial zinc . The genes encoding the urease of said organisms constitute a potential tool for the expression of recombinant urease products such as whole proteins or portions thereof.

A myriad of potentially useful urease preparations include peptides that are prepared from purified ureases (sources already mentioned) using physical and / or chemical cleavage procedures (i.e., CnBr) and / or enzymatic proteolysis using proteases, e.g. proteases V8, trypsin or others, or peptides synthesized chemically and having common epitopes with urease.

Another source of potentially useful epitopes are the epitopes identified due to their cross-reactivity with urease, which can be retrieved by anti-urease antibodies. These peptides may exist as natural peptides or may be produced by chemical synthesis. Further, such peptides may be produced by expression of a random recombinant oligonucleotide.

Another source of potentially useful epitopes is urease-like epitopes that arise from the production of anti-idiotypic anti-urease antibodies. Such anti-idiotypic antibodies produced by any immunocompetent host are obtained by immunizing that host with anti-urease antibodies to produce antibodies directed against anti-urease antibodies and having common structural homology to urease.

This discussion focuses on the use of natural urease produced by Helicobacter pylori (part B) as well as urease or its subunit or its constructs, which are already mentioned, which are capable of eliciting the desired protective immune response, and can also be produced using recombinant DNA techniques. which are well known to those skilled in the art. The efficacy of the individual formulations can be ascertained by routine administration using animal models, oral administration of a vaccine candidate, and exposure to a pathogen using a protocol essentially the same or similar to the procedure described below.

Tables 1 and 2 and Figures 1 to 5 describe the results obtained by oral immunization of mice with purified uricase from Helicobacter pylori. In this first experiment, administration of Helicobacter pylori antigen was performed orally by administering uricase from Helicobacter pylori to mice. Purification was carried out as described in Part A. Helicobacter pylori urease was adsorbed onto hydroxyapatite crystals, which is used as a carrier enhancing M-cell binding and absorption. Choleratoxin (Sigma) was added as a mucosal adjuvant. In this experiment, a group of six-week-old female BALB / c mice were orally immunized with 30 µg of purified Helicobacter pylori urease bound to 1 mg hydroxyapatite along with 10 µg of choleratoxin adjuvant on days 0, 7, 14 and 21. Mice were then exposed to 108 Helicobacter felis, on days 28 and 30. For comparison, similar, 6 week old female SPF-BALB / c mice were orally immunized with lysate from whole Helicobacter pylori (sonicate) with 10 µg of choleratoxin on days 0, 7, 14 and 21 Mice were exposed to Helicobacter felis on days 28 and 30. A sonicate of Helicobacter pylori was prepared by collecting Helicobacter pylori from cell cultures, centrifuging, dissolving the sediment in 0.9% NaCl, followed by sonication.

For control, female 6 week old SPF BALB / c mice were immunized with 10 µg choleratoxin and 1 mg hydroxyapatite on days 0, 7, 14, and 21. All mice were bred, immunized and exposed to the pathogen in parallel. All mice used for study were sacrificed for 35 days.

C-Oral immunization with recombinant subunits of urease from Helicobacter pylori

The genes encoding the Helicobacter pylori urease subunit A and B are obtained by cloning by polymerase chain reaction (PCR) according to standard procedures, based on previously published sequences [Clayton, Nucleic Acid Res., 1990, 18, 362]. These genes were inserted into a vector (named pEV 40), which was created for a high level of expression and easy purification of foreign genes in E. coli. Briefly, the foreign gene is inserted in the transcriptional direction of the thermosubstitutable promoter and in reading frame with a sequence of six histidines. The ampR gene is present in this vector for selection of transformants. Under appropriate temperature conditions, a protein supplemented at the N-terminus is obtained by a repeat of six histidines that allow one-step affinity purification on a nickel column. The two recombinant subunits (A and B) of urease from Helicobacter pylori were expressed separately in E. coli and purified on a nickel column up to 95% purity.

While it is preferred to use recombinant Helicobacter pylori urease as an antigenic material as described above, it is understood that any urease or uerase subunit obtained by recombinant techniques (e.g. fusion fusion) may be used as the antigenic material. protein), which has urease antigenic sites and which is capable of eliciting a protective immune response against Helicobacter infection. This means that the urease gene can be used to construct

Which has substantial homology to Helicobacter pylori urease, and which is effective in eliciting a cross-protective immune response against Helicobacteria. An example of such urease is Canavalia urease having about 70% homology to Helicobacter pylori urease, or Helicobacter felis urease having about 88% homology to Helicobacter pylori urease. Therefore, the invention is not limited to the use of Helicobacter pylori urease genes and gene products thereof, but encompasses the use of any recombinant urease or subunits thereof that are capable of eliciting a protective immune response in the host against Helicobacter infection. As recombinant antigen urease according to the invention, a recombinant urease having 70 to 95% homology, for example 80 to 90% homology, to urease from Helicobacter pylori can be used.

This discussion is directed to the use of recombinant A and B subunits of Helicobacter pylori urease which are produced by E. coli, but it is known that recombinant urease, or their subunits, or their constructs, which have already been mentioned, are capable of eliciting the desired protective immune response can be produced using other recombinant DNA techniques and other eukaryotic or prokaryotic expression vectors well known to those skilled in the art.

Tables 3, 4 and the bottom of Table 5 and Figure 6 describe the results obtained in experiments in which mice were orally immunized with recombinant Helicobacter pylori urease subunits produced in E. coli. In this experiment, Helicobacter pylori antigen was administered by orally administering the recombinant Helicobacter pylori urease A or B subunit, which is produced in E. coli and purified as described. The antigen was pre-bound to hydroxyapatite crystals, which are used as a carrier that enhances M-cell binding and antigen absorption. Choleratoxin (Sigma) is added as a mucosal adjuvant. A group of six week old female SPF-BALB / c mice was used in these experiments. Each mouse was orally immunized with 30 µg of recombinant A and B subunit of Helicobacter pylori urease conjugated to 1 mg hydroxyapatite, together with 10 µg of choleratoxin adjuvant. Immunization was performed on days 0, 8, 14 and 21. Mice were then exposed ^to Helicobacter felis 10 times three days on days 32, 34 and 36. For comparison, similar 6 week old female SPF-BALB / c mice, orally immunized with 30 µg of recombinant Helicobacter pylori urease B subunit, absorbed per 1 mg of hydroxyapatite, along with 10 µg of choleratoxin adjuvant. Immunization was performed on days 0, 8, 14 and 21. Mice were then exposed to 10 ⁸ Helicobacter felis three times on days 32, 34 and 36. All mice were bred, immunized, and pathogened in parallel. Mice used for study were sacrificed 48 days (12 days after meeting the pathogen) or 10 weeks after exposure to the pathogen.

D-Analysis of gastric biopsies, blood and intestinal secretions

The biopsy is taken from the stomach and blood is obtained from the heart. The intestines were collected and washed with 1 mM PMSF (Phenyl Methylsulfonyl Fluoride, Boehringer) in PBS (Phosphate Buffered Saline) to obtain intestinal secretions for ELISA analysis.

To evaluate protection against Helicobacter felis colonization, gastric biopsies from each animal are tested for Helicobacter felis using a rapid urease activity assay (Jatrox HP test, Rohm Pharma), following the supplier's guidelines. Briefly, the gastric biopsy is immersed in 0.5 ml of a mixture of urea and phenol red, which serves as a pH indicator, supplied directly by the supplier. Urease activity produces ammonia and bicarbonate from the urease and is accompanied by a colorimetric change due to the higher absorbance at 550 nm. Urease activity is quantified by spectrophotometric analysis.

To detect the presence of Helicobacter felis, gastric biopsies of all animals, including those used in the experiment described in Part B, are cultured on plates with BHI agarose supplemented as described. The presence of Helicobacter felis is confirmed by Gram staining and determination of urease activity after 3-10 incubations under microaerophilic conditions. Significant agreement was reached in the detection of Helicobacter felis cultures during the first set of experiments (see Table 3). In the experiment described in Part C, only tests to determine urease activity in gastric biopsies were performed to detect the presence of Helicobacter felis. The presence of Helicobacter felis was confirmed microscopically by two independent researchers, using two different staining methods (acridine orange and cresyl violet).

Blood samples are allowed to settle for three hours at room temperature, then serum is collected and allowed to freeze at -20 ° C until analysis time. The intestinal secretions are centrifuged for 5 minutes at 4 ° C to remove macroscopic particles and then stored frozen at -20 ° C. Determination of antihelicobacterial activity in the intestinal samples of each animal is performed by ELISA analysis according to standard procedures. Briefly, 96 well plates are coated with Helicobacter pylori sonicate and then saturated with 5% non-fat milk. Samples are serially diluted from 1: 1 to 1: 1000 dilution and incubated overnight on ELISA plates at 4 ° C. Biotinylated antibodies to IgG mice (for serum testing) and to IgA mice followed by streptavidin-horseradish peroxidase conjugate are used to determine antibody concentration.

The results of exposure of the animals to Helicobacter felis infection following immunization with purified Helicobacter pylori urease are shown in Tables 1 to 3 and Figures 1-4, and the results after exposure to Helicobacter felis infection following immunization with recombinant A and B subunits of Helicobacter pylori urease. are given in Tables 4 to 6 and Figures 5 to 6.

Table 1

Mouse Number immunization Urease test 12 h Gram staining immunoglobulins serum črev.í ig Ekre IgA Tg IgG 1 Urease + HP + H. felis 27 0 25 25Θ 2 Urease + HF 0 0 264 273 221 246 3 Urease + HF 0 0 84 44 318 354 4 Urease + HP H. felis 81 42 12 15 5 Urease + HF 0 0 98 137 126 234 6 Urease + HF ♦ 0 968 2093 31 22 7 Urease + HF 0 0 98 0 96 34 8 Urease + HF 0 0 247 1010 214 126 9 Urease + HF 0 0 nez. nez. 48 32 10 Urease + HF 0 0 with 0 124 99 11 urease 0 0 319 205 44 53 12 urease 0 0 14 0 86 87 13 urease 0 0 0 0 0 0

SK 280619 Β6

14 urease 0 0 0 0 43 61 15 urease 0 0 58 0 110 127 16 urease 0 0 140 63 21 37 17 urease 0 0 84 240 114 280 19 urease 0 0 before. nez. 93 148 19 urease 0 0 45 0 135 216 20 urease 0 0 261 197 161 261 21 ChT + HF 0 0 0 0 0 2 22 ChT + HF * H. felie 63 0 310 303 23 ChT + HF + you. felie 90 0 nez. nez. 24 ChT + HF - H. felie 31 0 150 192 25 ChT + HF - H. felis 197 250 250 440 26 ChT + HF ♦ H. felis 105 135 214 138 27 ChT + HF * H. felis 140 47 109 55 28 ChT + HF T 0 0 0 16 15 29 ChT + HF ♦ H. felis 0 0 0 0 30 ChT + HF + H. felis nez. before. nez. nez. 31 HP Sonate + HF H. felis 0 0 76 103 32 HP Sonate + HF * H. felis 77 0 11 33 33 HP Sonate + HF ⁺ H. felis 549 748 57 36 34 Hp sonicate + HF 0 0 660 153 180 266 35 HP Sonate + HF ⁺ H. felis 730 192 D 5 36 HP Sonate + HF ⁺ H. felis 32 0 5 64 37 HP Sonate + HF 0 0 400 400 312 1149 38 HP Sonate + HF * H. felis 1007 1360 149 26 39 Hp sonicate + HF 0 0 220 166 133 122 40 HP sonikát 0 0 873 1016 352 514 41 HP sonikát 0 0 72? 899 126 191 42 HP sonikát 0 0 109 68 44 83 43 HP sonikát 0 0 147 949 167 97 44 HP sonikát 0 0 845 1094 246 64 45 HP sonikát 0 0 1217 1198 210 157 46 HP sonikát 0 0 01 0 256 21B 47 1 HP sonikát 0 0 329 210 241 276 48 Hp sonicate 0 0 1049 737 197 211

In Table 1 describing the experiment described in Part B, the following abbreviations have the following meanings: "h" means hours, "Ig" means immunoglobulin, "nez" means "undetected", "urease + HF" means that the mouse has been immunized with urease ( hydroxyapatite and co-administered with choleratoxin) and then exposed to the pathogen Helicobacter felis, "urease" means that the mouse was immunized with urease (hydroxyapatite-bound and co-administered with choleratoxin) and not exposed to "ChT + HF" means that the mouse was immunized only with choleratoxin to control deception and exposed to Helicobacter felis, "HP sonicate + HF" means that the mouse is immunized with Helicobacter pylori sonicate, co-administered with choleratoxin, and exposed to Helicobacter felis, "HP means that the mouse is immunized with a Helicobacter pylori sonicate administered together with choleratoxin and is not exposed to the pathogen Helicobacter felis. In Table 1, the numbers giving the results of the antibody determination are derived from measurements of absorbance at a wavelength of 595 nm, multiplied by 1000. The background absorbance value, measured in the absence of antibodies, is subtracted from the result.

Table 2 shows the results of the experiment described in Part B, which are obtained from gastric biopsy urease tests and Gram staining of Helicobacter felis cultures. Mice with one or more Helicobacter felis colonization markers, including a urease assay, or Gram staining of cultures are defined as infected.

Table 2

immunization pathogen In infected individuals t protected individuals urease H. felis 3/10 (31%) 7/10 (69 *) * sonicate H.felis 6/9 (66%) 3/9 (33 V) » Ch.toxínom H.felis 9/10 (90 p) 1/10 (10 i)

* p ^_ 0.0198 (Fisher precision test) relative to choleratoxin-immunized control ** p = 0.303 (Fisher precision test) relative to choleratoxin-immunized control.

From the results shown in Tables 1 and 2, it is clear that oral immunization with urease from Helicobacter pylori gives statistically significant protection against the action of the pathogen Helicobacter felis, compared to protection obtained using either Helicobacter pylori sonicate or choleratoxin. From Table 2 it is clear that out of a total of 10 immunized animals only 3 are infected, compared to 6 animals that are immunized with choleratoxin. Table 2 shows that 70% of the animals are Helicobacter felis protected compared to 33% of the animals immunized with Helicobacter pylori sonicate and 10% of the animals immunized with choleratoxin and then exposed to Helicobacter felis. In other words, 90% of the control mice exposed to Helicobacter felis were infected with these pathogens, whereas in mice immunized with Helicobacter pylori urease 28 days prior to exposure to Helicobacter felis, the infection level was only 30%. This means a significant reduction in infection (p = 0.198 in Fisher's precision test) compared to control mice. In mice immunized orally with Helicobacter pylori sonicate, the level of infection is 67% (insignificant reduction over control). The protection obtained using Helicobacter pylori urease is unexpected and could not be expected from the results obtained using the Helicobacterpylori sonate.

Figure 1 graphically depicts the results of assays to determine serum antibody (IgG) and intestinal secretion (IgA) concentrations in mice that were not protected from infection after urease immunization. In Table 1, the mice are numbered 1, 4 and 6. These mice form group A. Figure 2 shows the antibody response in mice that are protected from infection after immunization (group B), i. j. mice 2, 3, 5, and 7-10. Figures 3 and 4 relate to the results obtained in mice 31-39. Figure 3 (group C) shows the antibody response of mice after immunization with Helicobacter pylori sonicate (mice number 31, 32). , 33, 35, 36 and 38) and Figure 4 (Group D) shows the antibody response of mice protected after immunization with Helicobacter pylori sonicate (mice number 34, 37 and 39). With respect to Figures 3 and 4, it is interesting that the antibody responses of IgA (but not IgG) are higher in mice that have protective immunity than in those that do not. Thus, the relationship between the protective and antibody responses of IgA can be assumed. Serum IgG has no such relationship. Mucosal IgA antibodies, but not serum IgG, are known to play a role in protection against bacterial intestinal infections [Parsonnet, J. Natl. Cancer Inst. 1991, 93, 640-643]

The results showing the correlation between the detection of Helicobacter felis in gastric biopsies by urease tests and detection by culture detection are shown in Table 3.

Table 3

Urease test + Ureázový test - Pretty Presence of H. felis + cultures 16 0 16 Presence of H. felis cultures - 2 30 32 Pretty 18 30 48

p <0.0001 in Fisher's precision test

Table 3 shows that there is a very significant correlation between the results of urease tests performed on gastric biopsies and the identification of Helicobacter felis infection. Urease tests were preferably used to diagnose Helicobacter felis infection in mice in further experiments due to their greater sensitivity. This approach allowed duplication of urease assays with larger gastric fragmentation in each mouse and further improved sensitivity of urease assays. The use of this method with a higher sensitivity has prevented the overestimation of the protection obtained by the test vaccine. Using a positive culture as the standard for infection, induced protection after urease immunization during the experiment described in Part B is as significant as in the combined use of the urease test and culture assays (p = 0.021 vs p = 0.019).

The results of the experiments described in Part C (recombinant urease subunits), which were obtained from urease tests from gastric biopsies, are shown in Tables 4, 5 and 6 and shown in Figure 6.

Table 4

immunization Mouse δ. Urease test infection ChT 20 0.49 T 21 0.31 22 0.62 * 23 0, 67 4 24 0.55 + 50 0, 50 4 51 0, 37 4 52 0.29 + 53 0.79 54 0.32 4 ureA + HAP + ChT 40 0.67 ¥ 41 0.48 ¥ 42 0.42 43 0.65 4 44 0.56 45 0.52 4 46 0.33 4 47 0.63 + 48 0.22 ♦ 49 0.37 + ureB + HAP + ChT 25 0.15 26 0.07 27 0.03 28 0, 64 4 29 0.13 30 0.02 31 0.66 + 32 0.00 ureA + HAP + ChT 68 0.00 69 0.07 70 0.42 4 71 0.00 72 0.00 ureB + HAP + ChT 73 0.37 + 74 0.00 - 75 0.37 76 0.00 - 77 0.00 - 78 0.00 - 79 0.39 + 80 0.00 - 81 0.37 * 82 0.00 -

In Table 4, the following abbreviations have the following meanings: "ChT" means choleratoxin, "UreA" means recombinant A subunit of Helicobacter pylori urease.

UreB means recombinant B subunit of urease from Helicobacter pylori, "HAP" refers to hydroxyapatite crystals. Mice 20-54 were sacrificed 12 days post-pathogen challenge and mice 68-82 after 10 weeks (106 days) post-pathogen challenge. The results of urease tests performed on gastric biopsies of each animal are expressed as absorbance values at 550 nm. The + signs indicate the final state of infection of individual animals with respect to the positivity or negativity of urease tests detecting the presence of Helicobacter felis. Absorbance values (at 550 nm) greater than 0.2 were considered positive.

Table 5

Protection measured in mice sacrificed 12 days after exposure to the pathogen

immunization pathogen % of infected individuals to the protected individuals A urease subunit H. felis 10/10 (99 4) 0/10 (1 ») fi subunit of urease H.felis 3/10 (30 υ 7/10 (69%) » Ch.toxínom H. felis 9/10 (91%) 0/10 (9 1)

* p = 0.0031 (Fisher precision test) relative to cholera toxin immunized control

Table 6

Protection measured in mice sacrificed 10 days after exposure to the pathogen

immunization pathogen In infected individuals % of protected individuals A urease subunit H. felis 1/5 (19 *) 4/5 (90t) B subunit of urease H. felis 4/10 (40t) 6/10 (61%) *

* p = 0.003 (Fisher precision test) relative to cholera toxin immunized control ** p = 0.01 (Fisher precision test) relative to choleratoxin immunized control.

From the results presented in Tables 4, 5 and 6, it is clear that statistically significant protection against the action of Helicobacter felis is achieved by oral immunization using the recombinant subunit of Helicobacter pylori urease as compared to protection obtained using either the recombinant A subunit. urease from Helicobacter pylori or choleratoxin. From Table 4, it can be seen that 12 days after exposure to the pathogen, of the 10 immunized animals from the group using the B subunit of urease, only 3 were found to be infected. By comparison, 10 of the 10 animals immunized with the urease A subunit were infected and 10 of the 10 immunized with choleratoxin were also infected. Table 4 shows that 70% of the animals were protected from Helicobacter felis treatment compared to 0% of the animals immunized with the A subunit of Helicobacter pylori urease and 0% of the animals immunized with choleratoxin and then exposed to Helicobacter felis. In other words, 100% of control mice exposed to Helicobacter felis were infected, whereas in mice immunized with the recombinant B subunit of Helicobacter pylori, the infection level was only 30%. This represents a significant reduction in infection (p = 0.0031, Fisher's precision test) compared to control mice.

SK 280619 Β6

The fact that the protection observed in Helicobacter pylori urease is partially conferred by immunization with the B subunit of urease and that the A subunit has no such effect was not expected from our experiments with purified urease. Therefore, defining the roles of the two structural subunits of urease in eliciting a protective immune response is entirely new. The protection induced by the use of recombinant B subunit urease which is enzymatically inactive demonstrates that a non-toxic form of urease can be used as an oral vaccine against Helicobacter infection. Furthermore, these results suggest that recognition of the active site is not necessary for protection because it is highly unlikely that the inactive urease B subunit induces antibodies that recognize and inhibit the catalytic site of the native urease.

Table 6 shows that from mice that were sacrificed 10 weeks after infection, 60% (6 mice out of 10) of animals immunized with urease B subunit and 80% (4 mice out of 5) of animals immunized with Helicobacter pylori B subunit urease were protected against Helicobacter felis infection. The fact that the protection obtained by immunization with the B subunit of urease persists for a long time and that immunization with the A subunit of urease induces only the protection that is disappearing compared to the protection induced by the B subunit of urease could not be expected from our purified urease experiments or other experiments performed. more likely. The fact that immunization with the B subunit of urease causes protection demonstrates definitively that recognition of the active site is not necessary for protective function. Figure 6 summarizes the results obtained after oral immunization with recombinant A and B urease subunits (described in Tables 5 and 6).

The invention further provides a composition of vaccines suitable for the prevention of Helicobacter infection. These compositions contain an effective amount of urease as an antigen, preferably a urease from Helicobacter pylori or a recombinant urease subunit from Helicobacter pylori, capable of eliciting a protective immune response in the host to Helicobacter infection. The compositions also include a pharmaceutically acceptable carrier or diluent.

The vaccines of the invention are administered in an amount readily determined by one skilled in the art. A suitable adult dose is in the range of 10 µg to 100 mg, e.g. from 50 pg to 50 mg. A similar dose range may be applied to children. Capsules from which the active ingredient is released in the small intestine may be used as a carrier system in humans, thereby protecting the antigen from the action of the acidic environment in the stomach and may also contain urease as an antigen in the insoluble form of alluvial proteins. The vaccine may be administered as a primary preventive agent to adults or children, as a secondary prevention following successful killing of Helicobacter pylori in an infected host, or as a therapeutic agent to induce an immune response in a susceptible host and thereby contribute to killing Helicobacter pylori infection.

A suitable mucosal adjuvant is choleratoxin, as mentioned previously. Further, muramyl dipeptide or derivatives thereof, non-toxic choleratoxin derivatives including its B subunit and / or conjugates or genetic engineering techniques prepared from urease as antigen and choleratoxin or its B subunit may be used. Other suitable routes of administration include biodegradable microcapsules or immune response stimulating complexes (ISCOM'S) or liposomes or simplified live vectors, prepared by genetic engineering techniques such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g. bluetongue. The amount of mucosal adjuvant used depends on its type. For example, when choleratoxin is used as a mucosal adjuvant, it is preferred to administer it in an amount of from 5 µg to 50 µg, e.g. 10 pg to 35 pg. Using microcapsules, the amount used depends on the amount of adjuvant that is contained in the microcapsule matrix to achieve the desired dose. Those skilled in the art will be able to determine this amount. Suitable carriers for the vaccine of the invention are capsules coated to release the contents in the small intestine and microspheres of polylactide-glycolide. Suitable solvents are 0.2 N NaHCO ₃ and / or saline.

Small Hydroxylated Calcium Phosphate (HCp) particles are particularly preferred as a carrier for Helicobacter pylori urease when applied to mucosal surfaces. Helicobacter pylori urease-hydroxylated calcium phosphate conjugate is believed to be transported by the epithelium where it elicits a poly-Ig immune response. It is preferred that the hydroxylated calcium phosphate is in the form of microparticles suitable for epithelial transport, especially for cells specialized for this purpose (M-cells). A preferred form of hydroxylated calcium phosphate is hydroxyapatite, a commercially available calcium phosphate Cai _O (PO ₄ ) ₆ (OH) ₂ .

Commercially available hydroxyapatite consists of seemingly viscous crystals that are chemically and physically analogous to inorganic hydroxyapatite in normal bone tissue. Hydroxyapatite digestion should therefore be safe, as evidenced by the existence of calcium and phosphorus-containing nutritional supplements derived from the bone mass that are produced for digestion. Commercially available hydroxyapatite (manufactured by CalBioChem) consists of crystals that vary widely in size. Crystals greater than 1 µm in length are unlikely to be absorbed by M cells. Therefore, for use according to the invention, the hydroxyapatite crystals are crushed into small, relatively equally sized crystalline fragments, e.g. sonication. It is preferred that the dimensions of the hydroxyapatite fragments present be in the range of about 0.01 to 0.0 µm. The success of fragmentation can be measured by either electron microscopy or light scattering, using standard techniques.

Preferred routes of administration of urease from Helicobacter pylori as an antigen are oral, nasal, rectal or ocular administration. Oral administration can deliver the antigen to another mucosal surface in the digestive tract, including intestinal mucosa.

The vaccines of the invention may be administered to the mucosal surface in the form of an aerosol, suspension, capsule and / or suppository. The mode of administration will be apparent to those skilled in the art.

The invention further provides passive immunization of a mammal, including a human, against Helicobacter infection. This is achieved by administering an effective amount of a specific urease specific antibody to the mucosal surface of the patient. It is preferred to use an effective amount of a monoclonal IgA antibody having specificity against Helicobacter pylori urease.

While it appears that Helicobacter pylori urease plays a role as an antigen of protective immunity, the invention also describes the use of Helicobacter pylori urease as a diagnostic reagent for measuring an immune response in persons receiving a urease-based vaccine or to determine whether is an individual immune or sensitive (hence needs vaccination). The invention further discloses the use of urease and specific urease antibodies for the construction of assays and diagnostic kits for determining the immunity against Helicobacter bacteria, for estimating susceptibility to these bacteria, and defining an immune response to vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be described in more detail in the following examples, which are not intended to limit the invention in any way.

A) Bacterial strains

Helicobacter felis was obtained by Mr. J. Fo (Cooperative Medicine Department, Massachusetts Institute of Technology, MIT, Boston, USA). Helicobacerpylori were isolated from patients with ulcer disease (CHUV, Lausanne, Switzerland).

B) Bacterial cultures

Liquid culture

The bacteria are cultured in liquid BHI medium (BioMerieux) containing 0.25% yeast extract (Difco) and 10% fetal calf serum (Inotech) supplemented with 0.4% supplement for selective growth of Campylobacter bacteria (Oxoid). The bacteria are incubated overnight under microaerophilic conditions at 37 ° C and then shaken at 37 ° C for 2-3 days.

Cultures on agarose plates

The bacteria are cultured on agarose plates containing BHI medium, 0.25% yeast extract and 5% sheep blood under microaerophilic conditions at 37 ° C for three days.

quantification

The amount of bacteria is determined by measuring the absorbance of the BHI solution at 660 nm (1 absorbance unit corresponds to 108 bacteria).

C) Preparation of sonicates

Helicobacter pylori was collected from 31 agar and blood plates and transferred to 0.15 M NaCl. They are then centrifuged for 5 minutes at 1400 g and 4 ° C. The sediment is then resuspended in 3 ml of NaCl and sonicated for 4 minutes. Protein levels were determined using the Bradford assay (BioRad Kid according to the supplier's instructions).

D) Conjugation of the immunogen with hydroxyapatite

Immunogen (urease or its subunit) is incubated for one hour with hydroxyapatite at 4 ° C. 1.0 mg of hydroxyapatite is used per 30 µg of immunogen per mouse. At the end of the incubation, 10 µg of choleratoxin is added to a final volume of 200 µl PBS.

Example 1

A) Extraction

Helicobacter pylori from 30 blood agar plates are transferred to 0.15 M NaCl on ice. The solution was centrifuged at 1400 xg and 4 ° C for 5 minutes. The sediment is resuspended in 20 ml H ₂ O and vortexed for 45 seconds (at maximum speed). The extract is then centrifuged at 6700 g and 4 ° C for 20 minutes. The supernatant was removed and the amount of proteins determined according to the above quantification and precipitated with 70% ammonium sulfate.

B) Purification of urease

The solution is subjected to chromatography on a column packed with scpharose CL-6B (Pharmacia) with PBS buffer, which is used as the mobile phase. The 22 fractions having urease activity were collected and dialyzed overnight at 4 ° C against 3 L PEB (20 nM phosphate buffer, pH = 7) and then subjected to Q-Sepharose flash column chromatography (Pharmacia). with PEB as the mobile phase. Fractions were eluted with a NaCl gradient of 0 to 500 nM. Ten of the collected fractions with strong urease activity were individually subjected to SDS gel electrophoresis followed by Coomassie staining. Six fractions containing two separate bands, corresponding to a molecular weight of 63 kDa and 28 kDa, were pooled and considered as purified urease.

Example 2 (see also Part B)

Mice used in immunization studies are first lightly anesthetized with ether and then gastrically immunized. The sonicate or purified urease, hydroxyapatite and choleratoxin are suspended in PBS and 200 µl of this mixture is delivered to the stomach of the respective mouse using a polyethylene tube attached to a syringe. We refer to this procedure as oral immunization.

Three immunization protocols were made and described below.

BI protocol - Vaccine purified urease

Female six-week-old BALB / c mice (20 animals) were orally immunized with 30 µg of purified Helicobacter pylori urease and 1 mg of hydroxyapatite and 10 µg of choleratoxin on days 0, 7, 14 and 21. Ten mice were then exposed to 5.10 ⁷ and 10 ^with Helicobacter felis from liquid culture on days 28 and 30.

Protocol B2 - Vaccination with Sonicate from Helicobacter

Female six-week-old BALB / c mice (20 animals) were orally immunized with 2 mg of Helicobacter pylori sonicate solution on days 0, 7, 14 and 21. Ten mice were then exposed to 5: 107 and 108 liquid culture Helicobacer felis, and on days 28 and 30.

Protocol B3 - Control

Female six-week-old BALB / c mice (20 animals) were orally immunized with 1 mg hydroxyapatite and 10 µg choleratoxin on days 0, 7, 14 and 21. Ten mice were then exposed to 5 x 10 7 and 10 8 liquid culture Helicobacer felis. on days 28 and 30.

On day 35, all mice were sacrificed and biopsies from the stomach, intestinal secretions and blood were collected.

Protection and evaluation of results

To evaluate the resulting protection, biopsies were tested and urease activity determined using the Jatrox HP assay (Rohm Pharma) following the supplier's instructions. The amount of urease was quantified by spectrophotometric measurement at 550 nm. Biopsies were also cultured in the presence of Helicobacter felis and the result was determined by Gram staining. Gastric cavity biopsies were homogenized and diluted (1: 10 and 1: 1000) in 0.15 M NaCl and then plated on blood-enriched agar plates and incubated under microaerophilic conditions at 37 ° C for 4-10 days.

SK 280619 Β6

ELISA

Determination of antibody titer in intestinal secretions and in blood is performed by ELISA analysis. ELISA analysis is performed as follows. 96-well polystyrene plates were coated with 1 µg per well of purified urease. The coating is carried out at 37 ° C and lasts for two hours. Non-specific binding sites are blocked with 5% milk powder in PBS buffer with 0.1% Tween at 37 ° C for 30 minutes. The plates are then washed once with PBS with 0.1% Tween. Blood samples are tested at a 1: 100 dilution and a 1: 1 intestinal secretion. Add 100 μΐ sample to each well on antigen coated plates. After two hours of incubation, the plates are washed three times with 0.1% Tween buffer. Total anti-mice biotinylated with goat antibodies and anti-mouse IgA, IgB and IgM biotinylated antibody (Amersham) are added (100 μΐ) at a 1: 500 dilution except for IgA added at a 1: 250 dilution and incubated for 1 hour at 37 ° C. C. The plates are then washed three times with PBS buffer with 0.1% Tween and then 100 µl of a 1: 1000 dilution of streptavidin-horseradish peroxidase in PBS buffer with 0.1% Tween is added. The plates are incubated for 30 minutes at 37 ° C. The plates are then washed three times and 50 μΐ of a solution of o-phenyl diamine in citrate buffer (1:50, pH = 5.0) with 1 μΐ / ml 30% H ₂ O _{2 is added} . The plates are incubated at room temperature for 20 minutes. Absorbance is measured at 495 nm in each well.

Example 3 (see also Part C)

Mice used in immunization studies are first lightly anesthetized with ether and then gastrically immunized. Then, 30 µg of the recombinant A and B subunit of Helicobacter pylori urease produced in E. coli bound to hydroxyapatite and supplemented with choleratoxin in PBS are suspended. 200 μΐ of this mixture is delivered to the stomach of the respective mouse using a polyethylene tubing connected by a syringe. We refer to this procedure as oral immunization.

Three immunization protocols were made and described below.

Protocol C1 - Vaccination of recombinant urease A subunit

Female 6-week-old BALB / c mice (10 animals) were orally immunized with 30 µg of purified recombinant A subunit of Helicobacter pylori urease and 1 mg of hydroxyapatite and 10 µg of choleratoxin on days 0, 8, 14 and 21. Ten mice were treated on days 32, 34 and 36 exposed to 10 ⁸ Helicobacter felis from liquid culture.

Protocol C2 - Vaccination with recombinant B subunit urease

Female six-week-old BALB / c mice (10 animals) were orally immunized with 30 µg of recombinant B subunit of Helicobacter pylori urease and 1 mg of hydroxyapatite and 10 µg of cholera toxin on days 0, 8, 14 and 21. Ten mice on day 32 , 34 and 36 exposed to 10 ⁸ Helicobacter felis from liquid culture.

Protocol C3 - Control

Female 6-week-old BALB / c mice (10 animals) were orally immunized with 1 mg hydroxyapatite and 10 µg choleratoxin on days 0, 8, 14 and 21. Ten mice were treated with 10 ⁸ Helicobacter felis from liquid on days 32, 34 and 36. culture. On day 42 or 106, mice were sacrificed and multiple biopsies were taken from the stomach.

Protection and evaluation of results

To evaluate the resulting protection, gastric cavity and gastric biopsy biopsies were tested by determining urease activity using the Jatrox HP assay (Rohm Pharma), following the supplier's instructions. The amount of urease was quantified by spectrophotometric measurement at 550 nm. The resulting absorbance value, valid for one mouse, was obtained as the sum of the values measured for cavity and corpus.

Claims (23)

A composition for inducing a protective or therapeutic immune response to a Helicobacter infection in a mammalian host, in particular for application to the mucosal surface of said mammal, comprising an immunologically effective amount of a polyamino acid preparation comprising a sufficient number of epitopes of an endogenous ureine bacterium Helicobacter for eliciting a protective immune response to infection by said bacterium. Composition according to claim 1, characterized in that it comprises intact urease obtained by purification from an organism with 70 to 95% homology to endogenous urease from Helicobacter. 3. A composition according to claim 1 comprising peptides with 70 to 95% homology to the enzymatically inactive portions of the Helicobacter urease amino acid sequence. A composition according to claim 1, characterized in that it comprises peptides that are not homologous to the urease amino acid sequence but which have epitopes capable of cross-reacting with Helicobacter urease. 5. A composition according to claim 1 comprising urease from Helicobacter pylori. A composition according to claim 1, characterized in that it comprises at least a subunit of urease from Helicobacter, which has or has no enzymatic activity. 7. A composition according to claim 1 comprising anti-idiotypic antibodies to urease from Helicobacter. 8. A composition according to claim 1, comprising peptides immunologically cross-reactive with urease from Helicobacter. Composition according to claims 3 and 8, characterized in that the peptides are obtained by chemical synthesis. 10. A composition according to claim 1 comprising urease antigens produced by recombinant DNA techniques having 70-95% homology to endogenous urease antigens from Helicobacter. 11. A composition according to claim 1 comprising subgenic urease fragments produced by recombinant techniques having 70-95% homology to the endogenous urease subunits of Helicobacter. 12. A composition according to claim 11 comprising subgenic urease fragments produced as genetic fusion proteins. 13. A composition according to claim 12, wherein the fusion proteins comprise choleratoxin subunits. 14. A composition according to claim 1 comprising a mucosal adjuvant. 15. The composition of claim 14, wherein the mucosal adjuvant is choleratoxin. 16. The composition of claim 1, further comprising hydroxylated calcium phosphate. 17. The composition of claim 16, wherein the hydroxylated calcium phosphate is hydroxyapatite. 18. The composition of claim 17, wherein the hydroxyapatite is in the form of particles suitable for epithelial transport. 19. A vaccine for eliciting a protective or therapeutic immune response to a Helicobacter infection in a mammal comprising a polyamino acid preparation having endogenous urease epitopes from Helicobacter on a pharmaceutically acceptable carrier or diluent. 20. The vaccine of claim 19, further comprising a mucosal adjuvant. A composition for providing passive protection to a mammal from Helicobacter-induced infection, in particular suitable for application to the mucosal surface of the mammal alone or after administration of the vaccine of claim 19 or 20, characterized in that it comprises an immunologically effective amount of an urease-specific IgA antibody. wherein the antibody has been produced in a mammalian host by immunization with urease which elicits a protective immune response to Helicobacter bacteria. 22. The composition of claim 21, wherein the antibody is an IgA antibody specific for urease from Helicobacter pylori. 23. A method for assessing the endogenous immune response of a mammal infected with Helicobacter, comprising determining in a sample taken from the gastrointestinal tract of the mammal the presence of an antibody reactive with epitopes of endogenous urease of said Helicobacter.